IgG-1 human monoclonal antibody reactive with an HIV-1 antigen and methods of use

ABSTRACT

The present invention provides a human-mouse myeloma analog, designated HMMA 2.11TG/O, which has been deposited with the American Type Culture Collection (ATCC) under Accession Number HB 9583. The invention also provides a hybridoma designated F 105, which also has been deposited with the ATCC under Accession Number HB 10363. The invention also concerns a monoclonal antibody-producing hybridoma produced by the fusion of the human-mouse myeloma analog and an antibody-producing cell. Other embodiments of the invention provide a method for producing a monoclonal antibody-producing hybridoma which comprises fusing the human-mouse myeloma analog with a human antibody-producing cell and a therapeutic method for treating a subject having a pathogen- or tumor-related disease which comprises administering to the subject a monoclonal antibody specific for the disease produced by the monoclonal antibody-producing hybridoma. In addition, a method of blocking binding of the human immunodeficiency virus and method of preventing infection of human cells by the human immunodeficiency virus are disclosed, as well as methods of detecting the virus or its antigen.

This application is a continuation of U.S. Ser. No. 08/252,545 filedJun. 1, 1994, now abandoned, which is a continuation of U.S. Ser. No.07/956,053, filed Oct. 2, 1992, now abandoned, which is a divisional ofU.S. Ser. No. 07/485,179, filed Feb. 26, 1990, now U.S. Pat. No.5,215,913, issued Jun. 1, 1993, which is a continuation-in-part of U.S.Ser. No. 07/126,594, filed Nov. 30, 1987, now abandoned, the contents ofwhich are hereby incorporated by reference.

The invention described herein was made in the course of work underGrant No. R01 CA 38687 from the National Cancer Institute, NationalInstitutes of Health, U.S. Department of Health and Human Services andGrant No. R01-AI 26926 from the National Institute of Health. The UnitedStates Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced byArabic numerals. Full citations for these references may be found at theend of the specification immediately preceding the claims. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The routine production of human monoclonal antibodies has been ofinterest since the construction of murine hybridomas which secretemurine monoclonal antibodies of predetermined specificity wereoriginally described (1).

While murine monoclonal antibodies provide valuable tools for the studyof biological processes, major limitations are apparent. First, thereare restricted number of antigens recognized by these antibodies (50).For example, antibodies directed to polymorphic determinants of the HLAand DR antigens have been difficult to identify (51). Moreover, it hasbeen almost impossible to identify specific human tumor-associatedantigens (51-57). Secondly, the pathogenesis of the autoimmunephenomenon in diseases such as diabetes require that the humanautoantibodies be defined (58). Finally, therapeutics using murinemonoclonal antibodies are restricted due to the formation of antimurineantibodies by the patients receiving the murine monoclonal antibodiesfor treatment (59-62). It is therefore likely that human monoclonalantibodies will provide major tools for the study of human neoplasia(9-14), autoimmune diseases (2-8), and infectious diseases (16-20), andwill serve as potential therapeutic and diagnostic agents for these andother illnesses.

To date, Epstein-Barr Virus (EBV) transformation of antibody-producinghuman B cells, selection of myeloma serum proteins, and fusion of bothmurine and human myeloma cell lines or analogs with antibody-producingcells have served as the only practical methods for obtaining humanmonoclonal antibodies. These methods, however, lack one or more of thefeatures which have made the routine production of murine monoclonalantibodies useful (21-26). While myeloma serum proteins have been usedby some investigators as sources of antibodies, this method is dependentupon the large scale screening of rare patients. Lack of reproducibilityand continual production, as well as restricted antigen specificities,limit the applicability of this method. EBV virus transformation ofantibody-producing B cells has provided the major source of humanmonoclonal antibodies reported in the literature. There are numerousinherent and methodological problems associated with the use of EBVtransformation as a technique for producing antibodies. First andforemost is the instability of monoclonal antibody production by thesecell lines (21). Because they have an extremely poor cloning efficiencyand unstable antibody secretion, only a few human monoclonalantibody-secreting cell lines have been maintained and have producedsufficient quantities of antibody for use in subsequent studies (3,11).Moreover, the low frequency and lack of specificity of EBVtransformation has necessitated selection methods designed to enhancethe recovery and transformation of antibody-secreting B cells (21, 29,30).

The development of human monoclonal antibodies by fusion of myeloma celllines or analogs with antibody-producing cells has been slowed by twomajor factors: 1) lack of an appropriate human fusion partner and 2)insufficiently available antigen-specific, human B cells. The presentlyavailable human fusion partners are lacking in important characteristicsnecessary for the production of monoclonal antibodies, i.e., efficientfusion, easy clonability of cell lines and fusion-resulting hybrids, andcontinuous secretion of large quantities of antibody by the hybrids.Without these characteristics, which are important features of murinefusion partners, it will be extremely difficult to obtain humanmonoclonal antibodies to many antigens. Human myeloma or lymphoblastoidcell lines have been used for fusion, but frequently these have either alow fusion efficiency, poor growth and cloning, or unstable secretion bythe resulting hybrids (6, 23, 31-33). For example, NSI, a murine myelomacell line, fuses with an efficiency of 1/10,000 with mouse spleen cells(66). Comparative fusion efficiency of LiCron HMY-2, SK007, UC729-6 orGM 1500 is between 1/500,000 and 1/1,000,000 with human cells (6, 33,67, 68). In addition, several of these cell lines, including derivativesof UC729-6 and LTR228, fuse poorly with normal peripheral bloodmononuclear cells (PBM). High fusion efficiency is particularlyimportant in a human system because of the relative rarity ofantibody-producing B cells, even in individuals undergoing programmedimmunization. In optimally tetanus immunized volunteers, as few as 1 outof 10,000 circulating B cells secrete anti-tetanus antibody (38). SinceB cells represent less than 10% of circulating PBM, large numbers wouldbe needed to obtain a single antibody-secreting hybrid. Directcomparisons of a number of human myeloma cell lines, mouse myeloma celllines, and human lymphoblastoid cell lines as human fusion partners havegenerally indicated fusion efficiencies on the order of 1/10⁵-10⁶ cells,with poor stability, and secretion between 100 ng and 10 μgm/ml inroutine cultures (6, 23, 31).

As an alternative to presently available human and murine cell linesused as fusion partners, a number of investigators have attempted toconstruct myeloma Analogs that might be superior for human monoclonalantibody production. Murine hybridization experiments have shown thatfusions between B cells with undifferentiated characteristics and Bcells with more differentiated characteristics result in the promotionof those differentiated characteristics in the hybrids (43, 44). Thus,Laskow, et al., and others, were able to promote the appearance ofphenotypic characteristics of a more differentiated B cell, specificallyincluding intact immunoglobulin production or secretion, by fusingundifferentiated B cells with a myeloma cell line (43-46). In theattempts to-construct a human myeloma analog that would retain thedesirable characteristics in the human fusion partner, it was theorizedthat the appropriate selection of cells for hybridization would resultin the sequential improvement of a series of constructed myeloma analogs(25, 26). These human myeloma analogs were constructed by the fusion ofa non-secreting human myeloma cell line with a variety of human cells atselected stages of differentiation. In these studies, while fusionefficiency was high and growth characteristics were excellent, stablesecretion of monoclonal immunoglobulin was obtained only from fusionswith established malignant human cell lines already committed tosecretion. Antibody secretion was rapidly lost by the cloned hybridomas.It is possible that the choice of the non-secreting human myeloma cellline as the basis for the series of constructed human myeloma analogsmay have had an impact on the ability of subsequently generated humanmyeloma analogs and hybridomas to support stable antibody production.

As an alternative to analogs formed by the fusion of human myeloma cellswith human cells, heterohybridomas have been constructed by the fusionof murine myeloma cells with human cells (34-37). Some investigators,including the present inventor, have constructed human-mouse myelomaanalogs by fusing murine myelomas with a variety of human cells. Themurine myelomas used for fusion derive principally from the MOPC21 cellline, developed by Potter and associates and adapted to in vitro growthby Horibata and Harris (27, 28, 39). This cell line and derivativesthereof are routinely used in the production of murine monoclonalantibodies as the fusion partner. Teng, et al., fused MOPC21 with thehuman cell line SK007 (34), Ostberg and Pursch fused it with a human Blymphocyte (37), and Foung, et al. fused normal peripheral bloodlymphocytes with a derivative of SP2, a murine myeloma hybrid (35).Carroll, et al., compared a number of these human-Rouse myeloma analogsfor fusion efficiency, immunoglobulin secretion and stability (36). Aheterohybridoma, K6H6/B5, constructed by fusion with NSI and human Blymphoma cells, was found to be superior to the other human-mousemyeloma analogs. This heterohybridoma has a fusion efficiency of 1/10⁵cells with 60% of the hybridomas secreting immunoglobulins.Immunoglobulin secretion by the hybrids was on the order of 2-3 μgm/ml.

Despite this effort, most heterohybridoma analogs have proven to haveunstable secretion (25, 26) or a poor fusion efficiency when compared tomurine myeloma cell lines. Since specific human antibody-producing cellsare rare in the peripheral blood, a higher fusion efficiency is adesirable feature of a human fusion partner (38).

The present invention provides a new human-mouse myelomas analog, whichhas been termed HMMA 2.11TG/0, a method oil constructing it and a methodof routinely using the human-mouse myeloma analog for the production ofhuman monoclonal antibodies. The HMMA 2.11TG/0 cell line has anextremely high fusion efficiency with normal PBM and EBV transformedPBM. It clones readily and, once cloned, stably secretes large amountsof human monoclonal antibody.

Human Immunodeficiency Virus 1 (HIV-1) infection represents a new andextremely serious health threat. The evolving epidemic has spread tonumerous risk groups in this country and new related viruses have nowappeared in Africa and other geographical areas (69-73). Patients withHIV-1 infection may develop a variety of directly related illnesses,including frank Acquired Immunodeficiency Syndrome (AIDS), AIDS RelatedComplex (ARC), encephalopathy, and AIDS related malignancies. Thesecomplications of HIV-1 infection are thought to derive in large partfrom a progressive and profound immunosuppression which occurs duringthe course of the illness, or to associated, possible, direct effects ofthe virus on specific organs, such as the brain (69-73). At the presenttime it is not known which patients infected with HIV-1 will go on tomanifest increasingly serious and morbid complications of the disease,and why some, but not all, individuals will undergo this progression. Itis felt that a profound depression of cellular immunity, as amanifestation of viral mediated destruction of T cells, may be involvedin this process. Inversion of the normal T4/T8 ratio, depletion oflymphocytes bearing the CD4 antigen, and lymphopenia in infectedindividuals are strongly correlated with progression to AIDS (74, 75).The preferential infection of T4 lymphocytes, syncytia formation anddeath of infected T4 cells, and the ability of some antibodies directedat the CD4 complex to prevent infection of cells by HIV-1, haveimplicated this population of T cells and the CD4 complex in thepathogenesis of this disease (72, 76-79).

While studies of immunodepression in this disease have concentrated onthe cellular arm of the immune response, the humoral immune system hasalso been profoundly effected by HIV-1 infection. Patients infected withHIV-1 have diminished responses to immunization with potent immunogenssuch as Keyhole Limpet Hemocyanin and Hepatitis B vaccines (80-82).Paradoxically patients also have serum hypergammaglobulinemia, possiblyas a result of chronic, non-specific, B cell stimulation. Several piecesof evidence support this contention. Isolated B cells from infectedindividuals are more likely to spontaneously secrete immunoglobulins aswell as specific antibodies, including antibodies to HIV-1 (80-82).These circulating B cells also appear activated on the basis of cellsurface phenotypic changes (83). In addition, circulating B cells fromHIV-1 infected patients are less likely to be transformed by exogenouslyadded Epstein Barr Virus (EBV), although spontaneous outgrowth of EBVtransformed B cells is higher than that seen in normals (82). BecauseEBV transformation preferentially occurs in non-activated B cells, thesedata support the notion that circulating B cells are chronicallyactivated (84). Taken together, these studies demonstrate thatalteration of the humoral immune response is a major occurrence in HIV-1infection.

The importance of the humoral immune response to HIV-1 in the in vivocontrol of the disease is controversial. Some epidemiologic studies ofrisk groups have suggested that the presence of serum antibody reactivewith the gp120 envelope protein of the HIV-1 virus, and capable ofneutralizing virus in infection assays, is correlated with lack ofdisease progression (85-87). Longitudinal studies of thalassemicpatients, infected via frequent blood transfusion, and of patientsinfected during treatment for curable malignancies support the notionthat neutralizing antibodies play a role in preventing the developmentof AIDS (85, 87). Conflicting evidence has also been presented tosuggest that these antibodies may have little role in preventingKaposi's Sarcoma, a manifestation of HIV-1 infection (88, 89). At leastone report has suggested that serum antibodies capable of blockingreverse transcriptase activity are also correlated with continued lackof disease progression (90). While the presence of neutralizingantibodies, and antibodies inhibiting reverse transcriptase activity,may be important in the in vivo control of this disease, they may alsorepresent epiphenomena of HIV-1 infection and their presence or absencemay be unrelated to the direct cause of further immunosuppression. Thismight pertain if, for example, viral infection occurred via cell-cellinteractions (91). Alternatively, genetic variation or drift in viralenvelope proteins may lead to escape from humoral immune control,although some epidemiologic studies have suggested that epitopesinvolved in viral binding and neutralization are frequently conservedacross isolates (92-95). A third mechanism for escape from control mightinvolve dysregulation of the humoral immune system leading to downregulation of antibody synthesis through the destruction of the CD4lymphocyte population (74, 82). It might be speculated that this processcould involve production of anti-idiotypic antibodies some of which maybind to T cells and contribute further to immunosuppression (96, 97).Thus, despite the tentative, speculative, and conflicting data regardingthe humoral immune response to HIV-1, it is of great importance that therelationship of the antibody response to HIV-1 and progression of thedisease be understood since there remains a real likelihood that thisresponse may significantly alter the course of infection.

Murine monoclonal antibodies reactive with either the CD4 complex orHIV-1 envelope proteins are capable of inhibiting infectivity of HIV-1in in vitro systems (77-79). These monoclonal antibodies are being usedto study idiotypic responses, CD4 attachment, and antigen binding siteson HIV-1 related proteins. For example, a murine monoclonalanti-idiotypic antibody, but not polyclonal rabbit anti-idiotypes,reactive with CD4 binding murine monoclonals reacts with HIV-1 envelopeproteins and inhibits cellular infection (79).

Despite the obvious utility of many of these antibodies and theiravailability for study, the precise determinants involved in the humanhumoral immune response remain unknown. In order to study both the humanhumoral response to HIV-1 infection, and the regulation of thisresponse, it is important to obtain the human equivalent of thecurrently available murine monoclonal antibodies. Human monoclonalantibodies to HIV-1 provide a series of uniform reagents that would beuseful in determining the precise epitopes involved in the immuneresponse to this virus, the idiotypic restrictions in the humoral immuneresponse, and the potentially important impact of anti-idiotypicregulation. Moreover, human monoclonal antibodies serve as usefuldiagnostic and therapeutic reagents in the evaluation and treatment ofthe disease. Therapeutic advantages of human monoclonal antibodies overmurine monoclonal antibodies include a decreased potential for directimmunization against the antibodies (98). In addition, anti-idiotypicantibodies binding to populations of normal T cells prove useful instudying the human immune response in general (99).

SUMMARY OF THE INVENTION

The present invention provides a human-mouse myeloma analog, designatedHMMA 2.11TG/O, which has been deposited with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure on Nov. 6, 1987. The human-mouse myeloma analog HMMA 2.11TG/O,was accorded ATCC Accession Number HB 9583.

The invention also concerns a monoclonal antibody-producing hybridomaproduced by the fusion of the human-mouse myeloma analog and anantibody-producing cell. Other embodiments oil the invention provide amethod for producing a monoclonal antibody-producing hybridoma whichcomprises fusing the human-mouse myeloma analog with a humanantibody-producing cell and a therapeutic method for treating a subjecthaving a pathogen- or tumor-related disease which comprisesadministering to the subject a monoclonal antibody specific for thedisease produced by the monoclonal antibody-producing hybridoma.

The invention provides a hybridoma deposited with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure on Feb. 23, 1990. The hybridoma was accorded ATCC AccessionNo. HB 10363 and designated F 105 and the human monoclonal antibodywhich it produces. The invention also concerns an anti-idiotypicantibody directed against this monoclonal antibody and methods of use.

This invention also provides a human monoclonal antibody directed to anepitope on human immunodeficiency virus (HIV) and capable of blockingthe binding of HIV to human cells and capable of preventing infection ofhuman cells by HIV.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: A representative photomicrograph of a chromosome preparationfrom the HMMA 2.11TG/0 cell line.

FIG. 1B: A representative photomicrograph of a chromosome preparationfrom the cloned antibody secreting hybridoma F3D₁,2F₆.

FIG. 1C: A representative photomicrograph of a chromosome preparationfrom the cloned antibody secreting hybridoma F5B₆A₆.

FIG. 2A: Shown are the results of indirect immunofluorescence of theF105 human monoclonal antibody, as compared to normal serum diluted1:100 (7%).

FIG. 2B: Shown are the results of indirect immunofluorescence of theF105 human monoclonal antibody, as compared to serum from an HIV-1infected patient HIV 24, diluted 1:100 (85%).

FIG. 2C: Shown are the results of indirect immunofluorescence of theF105 human monoclonal antibody, as compared to the F105 monoclonalantibody (54%).

FIG. 3: Shows the effect of human monoclonal antibody F 105 on viralbinding to HT-H9 cells.

FIG. 4: Shows that the F 105 human monoclonal antibody blocks HIV-1infectivity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a human-mouse myeloma analog designatedHMMA 2.11TG/O which has been deposited with the American Type CultureCollection (ATCC) Rockville, Md., U.S.A. 20852 under Accession Number HB9583 pursuant to the requirements of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure (Budapest Treaty).

The human-mouse myeloma analog of the present invention comprises hybridhuman-murine karyotypes and cell surface phenotypes and is produced bythe fusion of a mouse myeloma cell derived from MOPC21 and a human bonemarrow mononuclear cell. The mouse myeloma cell belongs to the mousemyeloma cell line designated P3x63Ag8.653, a non-secreting murinemyeloma cell line which is a mutant derivative of the cell line MOPC21.The human bone marrow mononuclear cell (BMMC) is obtained from humanswith a IgA/Kappa myeloma. The human donors are heavily treated withchemotherapeutic agents and are uremic and anemic prior to the obtainingof the BMMC by aspiration.

The analog resulting from the fusion of the BMMC and the mouse myelomacell is grown in the presence of thioguanine (particularly6-thioguanine) and ouabain and thus, is resistant to both ouabain andthioguanine. The resulting analog is also sensitive to mixtures ofhypoxanthine, aminopterin, and thymidine (HAT).

Another aspect of the present invention provides a monoclonalantibody-producing hybridoma produced by this fusion of the human-mousemyeloma analog and a human antibody-producing cell. In the preferredembodiments, the antibody-producing cell is a human peripheral bloodmononuclear cell (PBM), a mitogen stimulated PBM such as a PokeweedMitogen (PWM) or a phytohemagglutinin stimulated normal PBM (PHA(S)), oran Epstein-Barr Virus (EBV) transformed B cell. The human-mouse myelomaanalog described above has-an average fusion efficiency for growth ofantibody-secreting hybridomas of greater than 1 out of 25,000 fusedcells when fused with human PBM, mitogen stimulated PBM and EBVtransformed B cells. Especially useful antibody-producing hybridomas ofthe present invention are those hybridomas which produce monoclonalantibodies specific for human myelomonocytic leukemia, human acutelymphoblastic leukemia, human immunodeficiency virus (HIV), or humancolon carcinoma.

This invention further provides a human monoclonal antibody directed toan epitope on human immunodeficiency virus (HIV) and capable of blockingthe binding of HIV to human cells and capable preventing infection ofhuman cells by HIV. In one embodiment of the invention, the epitoperecognized by the human monoclonal antibody is the epitope recognized bya monoclonal antibody designated F 105. This invention also provides thehuman monoclonal antibody F 105. Monoclonal antibody F 105 is producedby a hybridoma also designated F 105 which has been deposited with theAmerican Type Culture Collection (ATCC) Rockville, Md., U.S.A. 20852under Accession Number HB10363 pursuant to the requirements of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure (Budapest Treaty).

Monoclonal antibody F 105 may be labelled with a detectable marker.Detectable markers useful in the practice of this invention are wellknown to those of ordinary skill in the art and may be, but are notlimited to radioisotopes, dyes or enzymes such as peroxidase or alkalinephosphatase. In addition, human monoclonal antibody F 105 may beconjugated with a cytotoxic agent.

This invention also concerns an anti-idiotypic antibody directed againstthe human monoclonal antibody F 105. This anti-idiotypic antibody mayalso be labelled with a detectable marker. Suitable detectable markersare well known to those of ordinary skill in the art and may be, but arenot limited to radioisotopes, dyes or enzymes such as peroxidase oralkaline phosphatase.

The anti-idiotypic antibody directed against human monoclonal antibody F105 is produced when an animal is injected with the F 105 antibody. Theanimal will then produce antibodies directed against the idiotypicdeterminants of the injected F 105 antibody (100).

Alternatively, the anti-idiotypic antibody directed against the humanmonoclonal antibody F 105 is produced by contacting lymphoid cells of ananimal with an effective-antibody raising amount of the antigen F 105;collecting the resulting lymphoid cells; fusing the collected lymphoidcells with myeloma cells to produce a series of hybridoma cells, each ofwhich produces a monoclonal antibody; screening the series of hybridomacells to identify those which secrete a monoclonal antibody capable ofbinding to the F 105 human monoclonal antibody; culturing the resultinghybridoma cell so identified and separately recovering theanti-idiotypic antibody produced by this cell (101). Animals which maybe used for the production of anti-idiotypic antibodies in either of thetwo above-identified methods include, but are not limited to humans,primates, mice, rats, or rabbits.

The invention also concerns a method for producing a monoclonalantibody-producing hybridoma which comprises fusing the human-mouseanalog with an antibody-producing cell, especially thoseantibody-producing cells listed hereinabove, and the monoclonal antibodywhich said hybridoma produces.

Another embodiment of the invention is a therapeutic method for treatinga subject having a pathogen- or tumor-related disease which comprisesadministering to the subject a monoclonal antibody specific for thedisease produced by the monoclonal antibody-producing hybridoma, whereinthe monoclonal antibody is capable of curing the disease or ofalleviating its symptoms. Preferably, the pathogen- or tumor-relateddisease is myelogenous leukemia, acute lymphoblastic leukemia, coloncarcinoma, acquired immunodeficiency syndrome, or human immunodeficiencyviral infection. The human-mouse myeloma analog of the present inventionand the monoclonal antibody-producing hybridomas produced therefrom arealso useful as research and diagnostic tools for the study of thesediseases.

The invention further concerns a method of blocking binding of the humanimmunodeficiency virus (HIV) to human cells and a method of preventinginfection of human cells by HIV which comprises contacting HIV with anamount of the human monoclonal antibody directed to an epitope on HIV,effective to block binding of HIV to human cells and preventinginfection of human sells by HIV.

A method of detecting in a sample the presence of HIV also is disclosedwhich comprises contacting a suitable sample with the monoclonalantibody F 105 so as to form an antibody-antigen complex between themonoclonal antibody and any HIV present in the sample and detecting thepresence of any complex so formed, thereby detecting in the sample thepresence of HIV. In one embodiment, the human monoclonal antibody F 105is labelled with a detectable marker. Suitable samples which are usefulin this method are, but are not limited to biological fluids from ahuman subject such as blood, serum, plasma, urine, nasal mucosaldischarge, oral mucosal discharge, vaginal mucosal discharge, analmucosal discharge and serosal fluids.

The method of detecting anti-HIV antibody also is provided by theinvention. The method comprises contacting a suitable sample with theanti-idiotypic antibody described hereinabove so as to form ananti-idiotypic antibody-anti-antibody complex between the anti-idiotypicantibody and any anti-HIV antibody in the sample and detecting thepresence of any complex so formed, thereby detecting in the sample thepresence of anti-HIV antibody. In one embodiment, the anti-idiotypicantibody is labelled with a detectable marker. Suitable samples whichare useful in this method are, but are not limited to, biological fluidsfrom a human subject such as blood, serum, plasma, urine, nasal mucosaldischarge, oral mucosal discharge, vaginal mucosal discharge, analmucosal discharge and serosal fluids.

A vaccine against human immunodeficiency virus (HIV) also is providedwhich comprises a human anti-idiotypic antibody described hereinabove inan amount effective to prevent HIV infection and a pharmaceuticallyacceptable carrier.

The invention further provides the DNA sequences which encodes for thevariable regions of the human monoclonal antibody F 105. The DNAsequences are isolated by first isolating and purifying the mRNAs whichencode for the F 105 heavy and light chain. cDNA copies are then madefrom these purified mRNAs to thereby provide the DNA sequences whichencode for the variable regions.

The present invention is further illustrated in the ExperimentalDetails, Results and Discussion sections which follow. These sectionsare set forth to aid in an understanding of the invention but are notintended to, and should not be constructed to, limit in any way theinvention as set forth in the claims which follow thereafter.

EXPERIMENTAL DETAILS Call Culture

Cell lines and established hybridomas were grown in Alpha-MEM, lackingnucleosides with the following additives: 1 mM sodium pyruvate, 2 mM1-glutamine, 1% (v/v) non-essential amino acids, 10% (v/v) fetal bovineserum (high cloning efficiency and growth promotion; GIBCO (GrandIsland, N.Y.), 0.22% (w/v) sodium bicarbonate, and 50 μgm/ml gentamycin.All other cell cultures were performed with the same media containing20% fetal bovine serum. Other additives were included as indicated.Cultures growing in flasks were sealed and maintained at 37° C. aftergassing with a 5% CO₂/air (v/v) mixture. Repeated gassings afterinitiation were performed as needed. Cultures in microtiter plates ormultiwells were incubated in a 5% CO₂ atmosphere at 37° C. in ahumidified incubator.

Cell Lines

The P3x63Ag8.653 cell line, a non-secreting murine myeloma cell line(39), was used for human-murine fusions (supplied by Dr. A. R.Frackelton). The B95-8 marmoset cell line (40) was used as a source ofEBV for cell transformation (supplied by Dr. H. Lazarus).

Volunteer and Patient Cells

PBM and BMMC were obtained by venipuncture or bone marrow aspiration,respectively, in preservative free heparin (O'Neal, Jones and Feldman;St. Louis, Mo.), and separated from contaminating cells by densitygradient separation as previously described (25, 26). If not usedimmediately cells were stored by cryopreservation in liquid nitrogenafter resuspension in media containing 10% dimethylsulfoxide. PBM fromvolunteers were obtained after immunization with tetanus toxoid atvarying time intervals.

Cell Fusion

Fusions were performed using a prepared 464 (w/v) solution ofpolyethylene glycol (PEG) 8000 (J T Baker Chemical Co., Phillipsberg,N.J.) in Puck's Saline G without calcium or magnesium (PSG/WO) (25). PEGwas autoclaved at 121° C. for 15 minutes and immediately mixed withPSG/WO warmed to 37° C. Sterile 1M sodium hydroxide was used to adjustthe pH to 8.0 and 4 ml aliquots were stored sterilely at 4° C., in glassvials, protected from light. The myeloma or myeloma analog was fused ina ratio of 2:1 with the other parental cells. Cells to be fused werepooled and washed twice with PSG/WO in a 50 ml polypropylene centrifugetube. After the final wash, the PSG/WO was decanted, the cell pellet wasresuspended in the residual PSG/WO, and 1.5 ml of PEG solution,previously warmed to 37° C., was slowly added, dropwise to the pelletwith frequent gentle mixing. The cells were allowed to incubate for 1minute and warm PSG/WO was added, dropwise to the pellet with frequentmixing. The first 1.0 ml was added over 1 minute, the second and thirdover 30 seconds each. Subsequently, a total of 20 ml was added to thecentrifuge tube. The cells were centrifuged at 400×G for 10 minutes, thePEG solution was decanted, and the cells were resuspended in culturemedia with hypoxanthine (1×10⁻⁴M), aminopterin (4×10⁻⁷M), and thymidine(1.6×10⁻⁵M) (HAT), (SIGMA, St. Louis, Mo.).

Fused cells were distributed in 96 well microtiter plates. For limitingdilutions, a set number of cells in 100 μl of media were placed in wellsin the first row (12 wells) of a microtiter plate in which all wellscontained 100 μl of media, and one half the volume transferredsequentially from one row to the next yielding serial two folddilutions. Fusion efficiency was calculated as previously described(25). The number of cells seeded into each well in any experiment wasbased on the maximum number of potential hybrids given a hypothetical1/1 fusion efficiency. The final volume in each well was 200 to 250 μl.In selected experiments 10 μM ouabain in 50 μl of media with HT wasadded to yield a 2 μM ouabain concentration in each well 24 hours afterfusion to prevent the growth of normal or transformed cells. Aconcentration of 2 μM ouabain was maintained in the wells for 1 week,after which routine feeding was performed. Fusions were fed at 4-7 dayintervals by removal of 100-150 μl of media and replacement with anequal volume of media containing hypoxanthine (2×10⁻⁴M) and thymidine(3.2×10⁻⁵M) (HT). After selection for expansion, cells were transferredto 24-well multiwells in media containing HT and were maintained in HTuntil they were passaged once in flasks.

Hybridomas were cloned by resuspension in media containing HT anddistributed in microtiter plates such that an average of 1 cell/100μl/well was obtained. Cloned cells were fed at weekly intervals withmedia lacking HT.

In Vitro Immunization and EBV Transformation

PBM from immunized volunteers were transformed to 24-well multiwells in2.0 ml of media containing 20% FBS and varying concentrations of tetanustoxoid (supplied by Wyeth Laboratories, Philadelphia, Pa.) prepared aspreviously described (25). As controls, wells were also prepared withoutadditives or with PWM (GIBCO, Grand Island, N.Y.,) diluted to a finalconcentration of 1:1000. Cells were fed initially on day 4 after thestart of the culture by removal of 1.8 ml of media and addition of freshmedia of the same amount, and thereafter at 4 to 10 day intervals byremoval of media and addition of fresh media. EBV transformation wasinitiated by removal of 1 ml of media from the cultures and addition of1 ml of a 1:5 dilution of a stock supernatant from the B95-8 cell linecollected after the method of Miller et al. (40). Wells to which EBV wasadded were monitored and fed weekly until continued cell growth andtransformation were evident. Growing cultures were transformed to 25 cm²flasks and expanded for fusion, continued culture and cryopreservation.

Preparation of IgG-1 Human Monoclonal Antibody

Peripheral Blood Mononuclear cells (PBM) from patients with seropositiveHIV-1 infection were isolated and transformed by Epstein Barr virus(EBV) in oligoclonal cultures. One of 915 transformants tested for theproduction of IgG cell surface reactive antibody to HIV-1 infected HT-H9cells was found to be positive. This EBV transformant was expanded andfused with the human fusion partner HMMA 2.11TG/O. Of 384 hybridomasscreened, 15 (4%) were producing antibody. Two hybridomas were clonedwith greater than 80% of tested clones producing the IgG humanmonoclonal antibody. F 105 human monoclonal antibody is an IgG-1immunoglobin.

Detection of Antibody and Immunoglobulin Secretion

Supernatants from test wells, or bulk cultures were tested forimmunoglobulin secretion using a microelisa assay as previouslydescribed (25, 26). In brief, test wells (Immulon 2; Dynatech,Alexandra, Va.) were coated with 100 μL of goat anti-humanimmunoglobulins (IgM, IgG, and IgA) (Cappell Laboratories,Cochraneville, Pa.) at 30 μgm/ml and incubated for at least 2 hours.Plates were then blocked with PSG with 2.5% PBS (v/v) (PSG 2.5%) for aminimum of two hours, washed twice with phosphate buffered saline (PBS)with 0.05% Tween® 20 (v/v) (PBS-tween) and twice with PBS and 100 μl oftest supernatant added. The wells were incubated for 2 hours, washed asabove, and 75 μl of peroxidase conjugated goat anti-humanimmunoglobulins (IgM, IgG and IgA) or specific peroxidase conjugatedgoat anti-human IgG, IgM or IgA (Tago, Inc., Burlingame, Calif.) diluted1:3000 in PSG2.5% were added and incubated for 2 hours. The wells werewashed 3 times with PBS-tween and 3 times with PBS after which 100 μl ofO-phenylenediamine in citrate buffer were added. Plates were read at5-45 minutes by observing a color change and scoring from negative to4⁺. Antibody to tetanus toxoid was detected using a similar assay exceptthat microelisa plates (Falcon Microtest III Assay Plates; BectonDickinson, Oxnard, Calif.) were coated with 100 μl of a tetanus toxoidsolution at 0.5 μgm/ml (25).

Cell Surface Immunofluorescence

Cell surface phenotypes were determined using both a direct and anindirect immunofluorescence method as previously described (25). In theindirect method, murine monoclonal antibodies specific for IgM, IgG,kappa light chains, lambda light chains, beta-2-microglobulin or Ia wereused with a supplied negative control (Coulter, Hialeah, Fla.).Fluorescein conjugated goat anti-mouse immunoglobulins (Tago,Burlingame, Calif.) was used to determine binding of the monoclonalantibodies. In the direct method, fluorescein conjugated goat anti-humanIgG, IgM, or IgA (Tago, Inc.) were used with fluorescein conjugated goatanti-mouse immunoglobulins as a negative control. Cell fluorescence wasdetermined using an Epics C cell sorter (Coulter, Hialeah, Fla.).

F 105 Indirect Immunofluorescence

The method by which antigen binding and infectivity was determined asfollows. HT-H9 cells may first be exposed to DEAE, or in thealternative, this step may be omitted. Virus, virus diluted with media,or virus diluted with F 105 supernatant and incubated for 30 minutes at37° C. is mixed with uninfected HT-H9 cells at 0.5 ml/10⁶ cells andallowed to incubate with these cells for 2 hours at 37° C. Followingcompletion of the 37° C. incubation period, the cells are washed withsterile media once, resuspended in PSG, and aliquoted at 1 millioncells/sample (for infectivity a sample is set aside for continued growthin regular media). The cells are then centrifuged for 5 minutes at1,000×g, the supernatant removed and individual samples are mixed with100 μl of normal serum or HIV⁺ serum diluted 1:200 or F 105 supernatantundiluted. The cells were then incubated for 30 minutes at 4° C., thenwashed with PSG (3-4 ml per wash). Fluorescein labeled, (F(AB′)₂) goatanti-human IgG antibodies, diluted 1:100, are then added (100 μl/sample)to each of the samples. The samples are then incubated for 30 minutes at40° C. and washed ×1. The cell pellet is resuspended in ½ ml of PBS and0.5% formaldehyde incubated for 30 minutes in the cold and then wereready for analysis on the cell sorter.

For infectivity, the cells are cultured and tested on days 4, 7 and 11for expression of surface viral antigens. The basic immunofluorescenceassay is as described above and will detect HIV antigens utilizingeither positive sera or F 105 on the surfaces of HT-H9 cells infectedwith HIV3B.

Quantitation of Antibody or Immunoglobulin Secretion

Spent culture media from flasks containing cloned growing hybridomacells were collected at 3-4 day intervals, centrifuged at 400 g for 30minutes, pooled and stored at 4° C. Immunoglobulin was concentrated 5-20fold by precipitation with saturated ammonium sulfate solution at 50%(v/v). For some experiments, antibody was further concentrated bycentrifugation over C50A membrane cones (Amicon Corporation, Danvers,Mass.). Ten microliters of the concentrated antibody, in PBS, were addedto the wells of a radial immunodiffusion plate with control referencestandards supplied (AccraAssay, ICN Pharmaceuticals, Lisle, Ill.). Theplates were evaluated 24-48 hours later. The quantity of immunoglobulinwas determined by comparison with standards.

Chromosome Analysis

Karyotypes were studied after preparation using a previously describedmethod (41).

EXPERIMENTAL RESULTS Construction of Human-Mouse Myeloma Analogs

Bone marrow mononuclear cells were obtained from a patient with anIgA/kappa myeloma and cryopreserved. Prior to the time of aspiration thepatient had been heavily treated with numerous chemotherapeutic agentsand was uremic and anemic. Eight gm/dl of monoclonal IgA immunoglobulinwere present in the serum. After recovery from liquid nitrogencryopreservation, 25×10⁶ bone marrow mononuclear cells fused with 50×10⁶murine myeloma cells and were seeded in microtiter wells at 0.25×10⁶fused cells/well. Ouabain was added to the wells after fusion. Threeweeks later 6 out of 96 wells demonstrated hybrid growth and 4 wereassayed for immunoglobulin secretion after transfer and further growthin multiwells. Three were found to secrete immunoglobulin.Immunoglobulin secretion was gradually lost over a period of 8 weeksafter fusion.

One hybridoma was selected on the basis of quantity and duration ofinitial secretion and cloned. Two clones from this hybridoma, designatedHMMA 2.11 and 2.12 were selected for further evaluation. The derivativecell lines were grown in the presence of increasing concentrations of6-thioguanine until growth of cells exposed to concentrations of 40μgm/ml of 6-thioguanine was equal to that of unexposed cell cultures.The cell lines were then grown in the presence of increasingconcentrations of ouabain until normal growth was observed atconcentration of 50 μm ouabain. The resulting cell lines were sensitiveto HAT.

One was found to be superior to the other on the basis of fusionefficiency (data not shown) and was evaluated, further.

This selected variant was termed MM 2.11TG/O and was deposited with theATCC under Accession Number HB 9583.

The cell line HMMA 2.11TG/O has a doubling time of 20-26 hours. Itsecretes neither IgG, IgM, nor IgA immunoglobulins detectable by ELISA.The hybrid derivation of the cell line was demonstrated by chromosomalanalysis (FIG. 1A) which revealed a mixed murine and human karyotypeamong the 77-89 (mean 85) chromosomes observed per cell. The cellsurface phenotype of the HMMA 2.11TG/O line also confirms the hybridnature of the cell line (Table I). The HMMA 2.11TG/O cells were found tolack Ia but retain beta-2-microglobulin. Surface IgA, IgM and IgG wereabsent as were kappa and lambda light chains. The surface phenotype ofthe original BMMC was strongly IgA and slightly IgM and Ia positive,while negative with IgG. These data indicate that the HMMA 2.11TG/O cellline is a mouse-human hybrid resulting from the fusion of the mousemyeloma P3x63Ag8.653 and cells from human myeloma containing bonemarrow.

TABLE I Immunofluorescent Analysis of the Cell Surface Phenotype of HMMA2.11TG/O and Donor Bone Marrow Nononucleer Cells Reactivity withcells^(a) Antigen HMMA 2.11TG/O BMMC Murine Monoclonal Antibodies IA − +B-2-M ++ N.D.^(b) I_(g)G − − I_(g)M − + Kappa − N.D. Lambda − N.D.Fluorescein Labled Goat Antibodies Murine I_(g)M + I_(g)G − − Human IgM− N.D. Human IgG − − Human IgA − +++ ^(a)− = <20%, + = 20-<50%, ++ =50-<75%, +++ = 75-100% ^(b)N.D. = not done

Production of Human Monoclonal Antibodies and Immunoglobulins

Fusions were performed with the HMMA 2.11TG/O cell line and fresh PBMfrom normal volunteers. As shown in Table II, fusion with PBM, and PWMstimulated PBM resulted in a relatively high recovery of hybrids. FusionHMMA 3.6 was performed with PBM stimulated with PWM for 4 days andseeded in a microtiter plate in limiting dilutions from 0.1×10⁶cells/well. At 3-4 weeks 43 wells were positive for hybrid growth. Thecalculated fusion efficiency was 1/20,050 cells. Thirty-three secretedimmunoglobulin. Two IgG secreting hybridomas and an IgM secretinghybridoma were cloned from wells seeded at the low dilutions of 25,000,1562 and 781 cells/well. Only 40% of the clones isolated from thesehybridomas were found to secrete immunoglobulins. These data suggestthat secretion may be unstable during the initial 4-6 weeks after fusionwith PWM stimulated PBM. The clone HMMA3.6 hybridomas continued tosecrete immunoglobulin until termination of the cultures 4 months later.

TABLE II Results of Fusions With the NMMA 2.11 TG/O Cell line RecipricalHybrids at Wells With Successfully Fusion^(a) Source of Cells^(b) FusionEfficiency^(c) 15,000 Cells/Well Ab or Ig^(d) Cloned/Cloned NMMA 3.6PBM-PWM-I 20,050 N.D. 33Ig 3/3 F₃ Tet₁B₅-EBV-I  5,060 N.D. 13Ab 1/3 F₅PBM-II 16,000 N.D. 3Ab 2/3 F₈ Tet₁B₁-EBV-I 13,360 56/96 12Ab 1/1 F₁₀Tet₃A₂-EBV-II 22,420 122/192 3Ab 1/1 F₁₁ Tet³⁻²B₂-EBV-II N.D. 295/3369Ab 2/3 F₁₂ Tet³⁻²B₄-EBV-II 25,650 117/192 1Ab^(e) N.D. CloningEfficiency Ab+ or Ig+ Clones Fusion 1 Cell/Well Positive Wells/WellsTested NMMA 3.6 20% 37/91 (40%) Ig F₃ 29% 9/63 (14%) Ab F₅ 32% 6/96 (6%)Ab F₈ 29% 19/44 (43%) Ab F₁₀ 11% 14/27 (32%) Ab F₁₁ 27% 14/51 (27%)Ab^(f) F₁₂ N.D. N.D. ^(a)Fusions F₁, F₂, F₄, F₆ and F₇ were performedwith other cell linen. ^(b)PWM = Pokeweed mitogen stimulated, EBV + ESVtransformed, TET = in vitro immunized, I, II = Volunteer source ofcells. ^(c)Number of cells fused/hybrid obtained ^(d)Ig =Immunoglobulin, Ab = Anti-tetanus Antibody ^(e)35 of 45 positive for IgMand 11 of a separate 45 positive for IgG Immunoglobulin secretion.^(f)See Table III

Hybridomas secreting monoclonal antibodies were directly obtainable froman appropriately immunized individual without further stimulation orselection. PBM from a volunteer immunized with tetanus toxoid wereobtained seven days after immunization and fused with HMMA 2.11TG/O at aratio of 2:1. A limiting dilution of fused cells was performed startingwith 0.2×10⁶ cells/well and three additional microtiter plates with70,000 fused cells/well were initiated. This fusion was termed F5. Theresulting fusion efficiency, as shown in Table II, was 1/16,000 fusedcells and all wells seeded at a density of 70,000 cells were: positivefor hybrid growth. From the entire F5 fusion, three wells were found tosecrete anti-tetanus IgM antibody and two were cloned. Over 50% of thewells contained anti-tetanus IgG antibodies (data not shown) but nonewere recovered on transfer to multiwells or cloning in this singleexperiment.

In order to demonstrate that it is possible to obtain monoclonalantibodies at a late date after in vitro immunization, in vivoimmunizations were performed with PBM from donors immunized with tetanustoxoid from 4 to 9 weeks previously. PBM cells were placed in wells of24 well plates at 1.8-2.5×10⁶ cells/well, and tetanus toxoid was addedto 1 μgm/ml to 0.1 ng/ml. In six experiments with three differentindividuals anti-tetanus antibody secretion was achieved without furtherstimulation and was detected by ELISA 8-12 days after the initiation ofthe cultures. PWM controls were positive for secretion whileunstimulated controls were negative (data not shown). Anti-tetanusantibody appeared in wells with various concentrations of tetanus toxoidin different experiments. Duplicate wells stimulated by tetanus toxoidwithin the range 50-100 ng and 1-5 ng/ml were positive in all theassays.

In five experiments EBV was added to all cultured wells at various timesafter the start of cultures. Transformation, as determined by increasingcell numbers and ability to expand in flasks was seen in all wells thatreceived EBV on days 8, 12, or 15 after in vitro immunization. Less than40% of wells showed growth if transformed on days 18 or 21. Anti-tetanusantibody secretion by transformed cells was detected in all wells thatwere initially positive for anti-tetanus antibody and subsequentlytransformed on days 12 and 15. An occasional well found to be negativeafter in vitro immunization became positive 2-3 weeks after EBVtransformation on days 12 and 15. Anti-tetanus antibody class waspredominantly IgM, with only approximately 20% oil antibody secretingwells secreting antibody of the IgG class.

Six separate polyclonal EBV transformed cell lines secretinganti-tetanus antibody from three in vitro immunizations from twoindividuals were selected for fusion. Generally polyclonal EBVtransformed cell lines were allowed to expand such that 7.5-10×10⁶ cellswere available, and 5×10⁶ were fused with HMMA 2.11TG/O cell line. Wherea fusion efficiency is indicated in Table II, limiting dilutions wereperformed. Where indicated, the remaining cells were distributed at15,000 cells/well into additional microtiter plates. Ouabain was addedto the fusions 24 hours later. Hybridoma formation was determined 21-28days after fusion at the time of initial testing for antibody secretion.The average fusion efficiency of HMMA 2.11TG/O with EBV transformed celllines was 1/16,600 cells. Seventy-two percent of wells seeded with15,000 fused cells/well had growing hybrids.

Hybridomas initially secreting anti-tetanus antibody were recovered andcloned from 4 to 6 fusions. Fusions F11 and F12 were performed with EBVtransformed cell lines secreting both IgG and IgM anti-tetanusantibodies. Cloned anti-tetanus antibody secreting hybridomas weresuccessfully recovered from the F3, F8, F10 and F11 fusions. Of the fiveseparate hybridomas cloned, three secrete IgM antibodies, and two, fromthe F11 fusion, secrete IgG antibodies. One fusion, F9 (data not shown,limiting dilution lost to contamination), resulted in no recoverableantibody secreting hybrids. Although the EBV transformed cell lines werecryopreserved after fusion, no additional fusions were performed.

To determine the frequency of immunoglobulin secreting hybridomas infusions with EBV transformed cell lines, hybridomas from the F9 and F12fusions were screened for IgG and IgM secretion. From the F9 fusion 28of 32 hybridomas tested secreted IgM, and 1 secreted IgG (91% totalsecretors). The F12 fusion has 35 of 45 hybridomas secreting IgM (78%)and 11 (24%) of an additional 45 secreting IgG. No attempt was made torecover immunoglobulin secreting clones from these fusions.

Cloning efficiencies of hybridomas from each fusion are shown in TableII and averaged 31%. Cloning was performed 4-6 weeks after fusion. Aspreviously noted, a significant fraction of wells containing clones werenegative for antibody secretion. To test the possibility that clonesnegative for antibody secretion might be secreting immunoglobulin,clones from two hybridomas of the F11 fusion were tested for IgG and IgMsecretion. An can be seen from Table III, lack of antibody secretion, incontrast to the HMMA 3.6 fusion, was not a result of the loss ofimmunoglobulin secretion since 90% of the clones secreted IgG, IgM orboth. Nonspecific immunoglobulin secretion by a large fraction of clonesmay be due to the presence oil multiple hybridomas in the primarycultures, or mutational loss of specificity. The secretion of both IgMand IgG in cloned populations is statistically consistent with havingseeded two cells in the same wells.

TABLE III Immunoglobulin and Anti-tetanus Antibody Secretion by PrimaryClones From Fusion F11 Number of Positive Clones Secreted ImmunoglobulinHybridoma Hybridoma or Antibody F11DE₂* F11CD₉* IgG Antibody 7 6 IgGImmunoglobulin 14  3 IgM Immunoglobulin 0 10  IgG + IgM Immunoglobulins0 2 None Detected 2 1 Total Screened 23 22

Phenotypic and Chromosomal Analysis

To demonstrate the hybrid derivation of antibody and immunoglobulinsecreting hybridomas, the surface phenotype of the parent cell lines andhybridomas were studied and the results are shown in Table I and IV. TheEBV transformed B cell lines bear typical cell surface antigenscharacteristic of the lineage and differentiation associated with theselines (42). At the tie of analysis at least two of these cell lines werepolyclonal as shown by the presence of both kappa and lambda positivecells. In contrast, the cloned hybrids are shown to lack Ia antigens,and beta-2-microglobulin is variably expressed. The cells from severalcloned hybridomas bear monoclonal IgM or IgG and a corresponding singlelight chain isotype on their surfaces while several IgG antibodyproducing cloned hybridomas have absent or reduced surface IgGexpression. Interestingly one is positive for IgA heavy chain.

Chromosomal preparations from two of the six cloned hybridomas studiesare shown in FIGS. 1B and 1C. Both human and murine chromosomes areclearly present in the preparations. In contrast to the HMMA 2.11TG/Ocell line which has 77-89 (mean 85) chromosomes/cell these clonedhybridomas have between 94 and 114 chromosomes/cell (mean 104).

TABLE IV Immunofluoruscent Analysis of the Cell Surface Phenotype ofHybridoma and EBV Transformed Cell Lines Antigens EBV Transformed ClonedHybridoma Cell Lines Cell Lines Tet³⁻²B₅ Tet³⁻²B₂ Tet₁B₅ F3D₁2F₆ F5C₉AD₂F8B₇B₈ F11CD₉B₅ F11DE₇F₄ HMMA3.61 Murine Monoclonal Antibodies Ia+++^(a) +++ +++ − − − − − − B-2-M +++ +++ +++ +++ ++ +++ ++ +++ +++ IgG− − − − − − + − ++ IgM + + +++ + ++ + − − − Kappa − + +++ + − + − − ++Lambda + + ++ − + − − − − Fluorescein Labeled Goat Antibodies MurineIgG, IgH − ND^(b) ND − − − − − ND Human IgM + ND ND +++ +++ +++ − − NDHuman IgG − ND ND − − − + − ND Human IgA − ND ND − − + − − ND ^(a)− =<20%, + = 20-<50%, ++ = 50-<75%, +++ = 75-100% ^(b)N.D. = not done

Quantity, Duration and Stability of Antibody Secretions

Antibody secretion was determined from cultures routinely kept in thelaboratory. Spent media from cultures of cloned hybridoma cell lineswere collected at 3 to 4 day intervals and pooled. Cells were generallyallowed to grow from 0.1×10⁶ to 0.5×10⁶ cells/ml. Supernatants wereconcentrated 10-20 fold and quantity was determined by radialimmunodiffusion. All concentrates were tested for reactivity withtetanus toxoid by ELISA and were found to be reactive at titers of1:40,000 or greater compared to 1:1500 for unconcentrated spent media.Hybridomas produced 8-42 μgm/ml of IgM (mean 22 μgm/ml) and 21-24μgms/ml (mean 22 μgm/ml) of IgG under these conditions (Table V).

TABLE V Antibody Secretion by Hybridomas and Subclones Cloned AntibodyQuantity Secreted Subclones Hybridoma Class (ugm/ml)^(a) SecretingAntibody^(c) F8B₇B₈ IgM 17 42% F8B₇C₁₂ IgM N.D.^(b) 50% F3D₁2F₆ IgM 4283% F5B₆A₆ IgM 21 100%  F5C₉AD₂ IgM 23 50% F10CE₇BF₉ IgM  8 100% F11CD₉B₅ IgG 21 95% F11DE₂F₄ IgG 22 80% ^(a)Secretion by HMMA 3.6H₉ = 24ugm/ml IgG ^(b)Not Done ^(c)% of wells positive for antibody, cloned at1 cell/well

Cloned hybridomas have continued to secrete monoclonal antibody ormonoclonal immunoglobulin for periods of 5-10 months after the originalcloning. At the present time, no continuously carried culture has lostantibody or immunoglobulin secretion after the initial cloning.Recloning of antibody-secreting hybrids revealed a broad range ofstability of secretion. Cloned hybridomas were recloned 4-12 weeks afterthe initial cloning and tested for anti-tetanus antibody secretion. Ascan be seen in Table V, 40-100% of new clones were secreting antibodywhen tested.

The F 105 Monoclonal Antibody

The results of indirect immunofluorescence are shown in FIG. 2. FIG. 2Ashows the reactivity of normal serum (diluted 1:100) with HIV3B infectedHT-H9 cells. Approximately 7% is considered a negative result. FIG. 2Bshows the reactivity of sera from patient HIV 24 (diluted 1:100).Eighty-five percent (85%) of the cells are reactive with this sera. FIG.2C represents the results with ones of several uncloned 105 hybridomas.The F 105 supernatant labels 54% of the cells. Because an IgG specificF(AB′)₂ goat antihuman FITC was used, this represents IgG antibodies.

F 105 reacts in a dot blot ELISA with HIV virus from supernatants of theHT-H9/HIV 3B infected cells. F 105 did not react with HIV-1 proteins ona commercial Western blot kit.

Table VI shows that F 105 blocks the binding and infection of DEAEprimed HT-H9 cells by HIV. The F 105 antigen was not detected on HT-H9cells after acute exposure to infectious virus but is detected withinseveral days post infection.

TABLE VI VIRUS F105 CELLS AB/SERA IF(1)* 1B − − HT-H9 NS 21 ± 13 21HIV24 19 ± 11 23 F105 11 ± 9  16 + − HT-H9 NS 20 ± 12 21 HIV24 50 ± 1093 F105 11 ± 10 79 + + HT-H9 NS 25 ± 16 28 HIV24 26 ± 12 33 F105 12 ± 1221 − − HIV_(3B) NS 12 ± 12 HIV24 85 ± 19 F105 63 ± 28 *The results areexpressed as per cent fluorescent cells using a goat-anti-human IgGF(AB′)₂ FITC labelled sera to develop the reaction. HTH9 cells wereexposed to DEAE and then to media, virus, or virus premixed withantibody. Exposure was for 0.5-2 hours and then tested for reactivitywith NS or HIV24 serum (1:50) or F105 supernatant (neat). HIV_(3B) wastested simultaneously. 1B represents the results with cells exposed inexperiment 1 and tested 4 days after experiment 1. Experiments are beingredone with fresh sera, mouse Ig blocking, and shorter incubation timesto reduce background.

In a second series of experiments, the results of which are set forth inTable VII below, it is shown that F 105 inhibits the binding of HIV-1 toHT-H9 cells.

TABLE VII F105 INHIBITS THE BINDING OF HIV-1 TO HT-H9 CELLS VIRUS F105CELLS AB/SERA IF(1)* 1F(2) IF(3) IF(4) − − HT-H9 NS 10 ± 3  — — 4 HIVPS10 ± 2  — — 3 F105 9 ± 3 — — 3 + − HT-H9 NS 7 ± 2 4 10 7 HIVPS 41 ± 2  513 42 F105  7 ± 2** 4 — 28 + + HT-H9 NS 7 ± 1 4 13 5 HIVPS 9 ± 2 4 10 18F105 — — — 9 − − HT-H9-HIV_(3B) NS 8 ± 2 14   7 7 HIVPS 55 ± 23 59  5967 F105 42 ± 19 54  43 48 *IF(1) Immediate IF after viral binding. Viralsupernatant and F105 or media were mixed 1:1 and incubated for 30minutes and then added to cells for 2 hours. These are the average of3-4 experiments with standard deviations. IF(2), IF(3), and IF(4) referto cell assays performed 3-4 days, 6-7 days, and 10-11 days afterinfection respectively and are the average of 2 experiments. **F105 isnegative immediately after viral binding, see additional data. HIVPS andNS are sera from HIV positive patients and normal volunteersrespectively. Sera are used at a 1:200 dilution.

The human monoclonal antibody F 105 reacts with a conformationallydetermined epitope on HIV-1 virions to prevent viral binding andinfection. (See FIGS. 3 and 4.)

Experimental Discussion

The present invention provides a method for the construction of ahuman-souse myeloma analog for the production of human monoclonalantibodies. The human-mouse myeloma analog was constructed by fusion ofbone marrow mononuclear cells from a patient with IgA myeloma and anon-secreting variant, P3x63Ag8.653, of the mouse myeloma cell lineMOPC21 (39). The nonsecreting, cloned, mutant hybridoma, HMMA 2.11TG/Ois resistant to 6-thioguanine and ouabain and sensitive to HAT. The cellline, HMMA 2.11TG/O, has a high fusion efficiency with peripheral bloodmononuclear cells, Pokeweed Mitogen stimulated peripheral bloodmononuclear cells, and EBV transformed B cell lines. The cloningefficiency of the second generation hybridomas is high and does notrequire the presence of feeder cells. Seven separate hybridomas fromfive fusions secreting anti-tetanus monoclonal antibodies were cloned.Five of these are of the IgM class and two are of the IgG class.Antibody and immunoglobulin secretion is stable and secretion has beenmaintained for 5-10 months without recloning. Secretion of both classesof antibody is greater than 8 μgm/ml and as high as 42 μgm/ml in routineculture. Chromosomal analysis reveals a hybrid karyotype in both theHMMA 2.11TG/O cell line and subsequent second generation hybridoma.

In the present invention, the cell line P3x63Ag8.653 was used as themurine fusion partner because it was a non-producing derivative ofMOPC21, and is a cell line of proven efficacy in the production ofmurine monoclonal antibodies (39). As the human fusion partner, it wasreasoned that the most differentiated cell available would be foundamong the bone marrow cells from a patient with multiple myeloma.Moreover, an IgA myeloma was chosen so that, should the analog secreteimmunoglobulin, it would be readily distinguishable from the moredesirable IgG and IgM antibodies. While the original fusion gave resultsconsistent with those of other researchers performing human-mousefusions, e.g. low fusion efficiency and rapid loss of secretion, theresulting non-secreting hybrid cell line has phenotypic characteristicsof both the murine and human parental cells and fuses much more readilywith human B cells. Moreover, the secretion of human immunoglobulin andantibody by the resulting second generation hybrids is consistent withthat seen by murine myeloma cell lines and hybrids (43). Thus, thefusion of the murine myeloma cells with human cells derived from apatient with myeloma has resulted in a human-mouse myeloma analog withhuman fusion characteristics similar to those seen in murine monoclonalantibody methods.

Since the second important feature of the murine monoclonal antibodytechnology is the ready availability of larger numbers ofantibody-producing B cells after programmed immunization, it isimportant that the HMMA 2.11TG/O cell line be able to fuse with PBMcells from an appropriately immunized donor and readily give rise toantibody secreting hybridomas that are recoverable as cloned secretingcell lines without special selection techniques (47). Nonetheless, manyantibodies to specific antigens of interest may not be as readilyobtainable, either because in vivo immunization has occurred at someremote, or nonoptimal time in the past, or is not feasible. Thus, it isimportant to be able to stimulate and expand the antibody-secreting Bcell population for fusion (21). In vitro immunization has beensuccessfully used for the production of a secondary immune response totetanus (48), viral antigens (15, 21), and parasite antigens (49), amongothers. Numerous polyclonal antibody-secreting EBV transformed B celllines have been obtained by transformation of peripheral blood, draininglymph nodes, and la vitro immunized B cells (21). The experimental dataset forth hereinabove demonstrates that it is possible to immunizeroutinely in vitro, EBV transform, and expand antibody-producing B cellswhich can subsequently be fused with the HMMA 2.11TG/O cell line with ahigh likelihood of recovery of clonable antibody-producing hybrids.Thus, fusions can be performed with already available EBV transformed Blymphoblastoid cell lines secreting known antibody, PBM from optimallyimmunized volunteers or patients, stimulated PBM, or in vitro immunizedand EBV transformed cells.

The data reported herein demonstrate that the human-mouse myelomaanalog, HMMA 2.11TG/O, is a superior fusion partner for the productionof human monoclonal antibodies. Antibody-producing hybridomas can berecovered after fusion with B cells both directly from peripheral bloodand EBV transformed polyclonal cell lines. This cell line should bevaluable in the study of a variety of human diseases.

REFERENCES

1. Kohler, G., Milstein, C., 1975, Continuous Cultures of Fused CellsSecreting Antibody of Predefined Specificity, Nature, 256:495.

2. Steinitz, M., Izak, G., Cohen, S., Ehrenfeld, M., Flechner, I., 1980,Continuous Production of Monoclonal Rheumatoid Factor by EBV-TransformedLymphocytes, 1980, Nature, 287:443.

3. Sasaki, T., Endo, F., Mikami, M., Sekiguchi, Y., Tada, K., Ono, Y.,Ishida, N., Yoshinage, K., 1984, Establishment of Human MonoclonalAnti-DNA Antibody Producing Cell Lines, J. Immunol. Meth., 72:157.

4. Gaskin, F., Kingsley, B., Fu, S. M., 1987, Autoantibodies toNeurofibrillary Tangles and Brain Tissue in Alzheimer's Disease:Establishment of Epstein-Barr Virus Transformed Antibody-Producing CellLines, J. Exp. Med., 165:245.

5. Eisenbarth, G. S., Linnenbach, A., Jackson, R., Scearce, R., Croce,C. M., 1982, Human Hybridomas Secreting Anti-Islet Autoantibodies,Nature, 300:264.

6. Satoh, J., Prabhaker, B. S., Haspel, N. V., Ginsberg-Fellner, F.,Notkins, A. L., 1983, Human Monoclonal Autoantibodies that React withMultiple Endocrine Organs, N. Engl. J. Med., 309:217.

7. Valente, W. A., Vitti, P., Yavin, Z., Yavin, B., Rotella, C. M.,Grollman, E. F., Toccafondi, R. S., Kohn, L. D., 1982, MonoclonalAntibodies to the Thyrotropin Receptor: Stimulating and BlockingAntibodies Derived from the Lymphocytes of Patients with Graves Disease,Proc. Natl. Acad. Sci. (USA), 79:6680.

8. Alpert, S. D., Turek, P. J., Foung, S. K. H., Engleman, E. G., 1987,Human Monclonal Anti-T Cell Antibody from a Patient with JuvenileRheumatoid Arthritis, J. Immunol., 138:104.

9. Wunderlich, D., Teramoto, Y. A., Alford, C., Schlom, J., 1981, TheUse of Lymphocytes from Auxillary Lymph Nodes of Mastectomy Patients toGenerate Human Monoclonal Antibodies, Eur. J. Cancer, 17:719.

10. Watson, D. B., Burns, G. F., Mackay, I. R., 1983, In vitro Growth ofB Lymphocytes Infiltrating Human Melanoma Tissue by Transformation withEBV: Evidence for Secretion of Anti-Melanoma Antibodies by SomeTransformed Cells, J. Immunol., 130:2442.

11. Irie, R. F., Sze, L. L., Saxton, R. E., 1982, Human Antibody toOFA-I, a Tumor Antigen, Produced In vitro by Epstein-BarrVirus-Transformed Human B-Lymphoid Cell Lines, Proc. Natl. Acad. Sci.(USA), 79:5666.

12. Andreasen, R. B., Olsson, L., 1986, Antibody-Producing Human-HumanHybridomas; III. Derivation and Characterization of Two Antibodies withSpecificity for Human Myeloid Cells, J. Immunol. 137:1083.

13. Shoenfeld, Y., Ammon, B., Tal, R., Smordinsky, N. I., Lavie, G.,Mor, C., Schteren, S., Mammon, Z., Pinkhas, J., Keydar, T., 1987, HumanMonoclonal Antibodies Derived from Lymph Nodes of a Patient with BreastCarcinoma React with MuMTV Polypeptides, Cancer, 59:43.

14. Sikora, K., Alderson, T., Ellis, J., Phillips. J., Watson, J., 1983,Human Hybridomas from Patients with Malignant Disease, Br. J. Cancer,47:135.

15. Crawford, D. H., Callard, R. E., Muggeridge, M. I., Mitchell, D. M.,Zenders, E. D., Beverley, P. C. L., 1983, Production of Human MonoclonalAntibody to X31 Influenza, Virus Nucleoprotein, J. Gen. Virol., 64:697.

16. Zurawski, V.R., Spedden, S. E., Black, P. H., Haber, E., 1978,Clones of Human Lymphoblastoid Cell Lines Producing Antibody to TetanusToxoid, Curr. Top. Microbial. Immunol., 81:152.

17. Rosen, A., Persson, K., Klein, G., 1983, Human Monoclonal Antibodiesto a Genus-Specific Chlamydia Antigen, Produced by EBV-Transformed BCells, J. Immunol., 130:2899.

18. Gigliotti, F., Insel, R. A., 1982, Protective Human HybridomaAntibody to Tetanus Toxin, J. Clin. Invest., 70:1306.

19. Croce, C. M., Linnenbach, A., Hall, W., Steplewski, Z., Koprowski,H., 1980, Production of Human Hybridomas Secreting Antibodies to MeaslesVirus, Nature, 288:488.

20. Larrick, J. W., Truitt, K. E., Raubitschek, A. A., Senyk, G., Wang,J. C. N., 1983, Characterization of Human Hybridomas Secreting Antibodyto Tetanus Toxoid, Proc. Natl. Acad. Sci. (USA), 80:6376.

21. Crawford, D. H., 1986, Use of Virus to Prepare Human-DerivedMonoclonal Antibodies, In: The Epstein-Barr Virus Recent Advances, Ed:Epstein, M. A. Achong, B. G., John Wiley, and Sons, New York, 251.

22. Kabat, E. A., Nickerson, K. G., Liao, J. Grossbard, L., Osserman, E.F., Glickman, E., Chess, L., Robbins, J. B., Schneerson, R., Yang, Y.,1986, A Human Monoclonal Macroglobulin with Specificity fora(2-8)-Linked Poly-N-Acetyl-Neuraminic Acid, The Capsular Polysaccharideof Group B Meningococci and Escherichia Coli K1, which Crossreacts withPolynucleotides and with Denatured DNA, J. Exp. Med., 164:642.

23. Olsson, L., Kronstrom, H., Cambon-De Mouzon, A., Honsik, C.,Jakobsen, B., 1983, Antibody Producing Human-Human Hybridomas, I.Technical Aspects, J. Immunol. Meth., 61:17.

24. Schwaber, J., Cohen, E. P., 1973, Human X Mouse Somatic Cell HybridClone Secreting Immunoglobulins of Both Patental Types, Nature, 244:444.

25. Posner, M. R., Schlossian, S. F., Lazarus, H., 1983, Novel Approachto the Construction of Human “Myeloma Analogues” for the Production ofHuman Monoclonal Antibodies, Hybridoma, 2;369.

26. Schwaber, J. F., Posner, M. R., Schlossian, S. F., Lazarus, H.,1984, Human-Human Hybrids Secreting Pneumococcal Antibodies, HumanImmunol,. 9:137.

27. Potter, M. O., 1972, Immunoglobulin-Producing Tumors and MyelomaProteins of Mice, Physiol. Rev., 52:631.

28. Horibata, K., Harris, A. W., 1970, Mouse Myelomas and Lymphomas inCulture, Exp. Cell Res. 60:61.

29. Casali, P., Inghirami, G., Nakamurta, M., Davies, T. F., Notkins, A.L., 1986, Human Monoclonals from Antigen-Specific Selection of BLymphocytes and Transformation by EBV, Science, 234:476.

30. Steinitz, M., Koskimies, S., Klein, G., Makela, O., 1978,Establishment of Specific Antibody Producing Human Lines by AntigenPreselection and EBV-Transformation, Curr. Top. Microbiol. Immunol.,81:156.

31. Cote, R. J., Morrissey, D. M., Houghton, A. N., Beattie, E. J.,Oettgen, H. F., Old, L. J., 1983, Generation of Human MonoclonalAntibodies Reactive with Cellular Antigens, Proc. Natl. Acad. Sci.(USA), 80:2026.

32. Brodin, T. , Olsson, L., Sjorgren, H., 1983, Cloning of HumanHybridoma, Myeloma, and Lymphoma Cell Lines Using Enriched HumanMonocytes as Feeder Layer, J. Immunol. Meth., 60:1.

33. Glassy, M. C., Handley, H. N., Hagiwara, H., Royston, I., 1983, UC729-6, A Human Lympholastoid B-Cell Line Useful for GeneratingAntibody-Secreting Human-Human Hybridomas, Proc. Natl. Acad. Sci. (USA),80:6327.

34. Teng, N. N. H., Lam, K. S., Riera, F. C., Kaplan, H. S., 1983,Construction and Testing of Mouse-Human Heteromyelomas for HumanMonoclonal Antibody Production, Proc. Natl. Acad. Sci. (USA), 80:7308.

35. Fuong, S. K. H., Perkins, S., Raubitschek, A., Larrick, J., Lizak,G., Fishwild, D., Engleman, E. G., Grumet, F. C., 1984, Rescue of HumanMonoclonal Antibody Production from an EBV-Transformed B Cell Line byFusion with a Human-Mouse Hybridoma, J. Immunol. Meth., 70:83.

36. Carroll, W. L., Thielmans, K., Dilley, J., Levy, R., 1986, Mouse XHuman Heterohybridomas As Fusion Partners with Human B Cell Tumors, J.Immunol. Meth., 89:61.

37. Ostberg. L., Pursch, E., 1983, Human X (Mouse X Human) HybridomasStably Producing Human Antibodies, Hybridomas, 2:361.

38. Stevens, R. H., Macy, E., Morrow, C., Saxon, A., 1979,Characterization of a Circulating Subpopulation of SpontaneousAntitetanus Toxoid Antibody Producing B Cells Following in Vivo BoosterImmunization, J. Immunol., 122:2498.

39. Kearney, J. P., Radbruch, A., Liesgang, B., Rajewsky, K., 1979, ANew Mouse Myeloma Cell Line that has Lost Immunoglobulin Expression butPermits the Construction of Antibody-Secreting Hybrid Cell Lines, J.Immunol, 123:1548.

40. Miller, G., Lipman, M., 1973, Release of Infectious Epstein-BarrVirus by transformed Marmoset Leukocytes, Proc. Natl. Acad. Sci. (USA),70:190.

41. Weitberg, A. B., Weitzman, S. A., Destrempes, M., Latt, S. A.,Stossel, T. P., 1983, Stimulated Human Phagocytes Produce CytoogeneticChanges in Cultured Mammalian Cells, New Eng. J. Med., 308:26.

42. Halper, J., Fu, S. M., Wang, C. Y., Winchester, R., Kunkel, H. G.,1978, Patterns of Expression of Human “Ia-Like” Antigens During theTerminal Stages of B Cell Development, J. Immunol., 120:1480.

43. Laskov, R., Kim, K. J., Asofsky, 1979, Induction of AmplifiedSynthesis and Secretion of IgM by Fusion of Murine B Lymphoma withMyeloma Cells, Proc. Natl. Acad. Sci. (USA), 76:915.

44. Laskov, R., Kim, J. K., Kanellpoulos-Langevin, C., Asofsky, R.,1980, Extinction of B-Cell Surface Differentiation Markers in HybridsBetween Murine B-Lymphoma and Myeloma Cells, Cell Immunol., 55:251.

45. Riley, S. C., Brock, E. J., Kuehl, W. M., 1981, Induction of LightChain Expression in a Pre-B Cell Line by Fusion to Myeloma Cells,Nature, 289:804.

46. Hamano, T., Kin, K. J., Lieserson, W. M., Asofsky, R., 1982,Establishment of a B Cell Hybridoma with B Cell Surface Antigens, J.Immunol., 129:1403.

47. Posner, M. R., Berkman, R., Fife, J., Lazarus, H., 1984, OptimalConditions for Obtaining Human Monoclonal Antibodies After Immunizationwith Tetanus, Clin. Res., 32:355A.

48. Volkman, D. J., Allyn, S. P., Fauci, A. S., 1982, Antigen-Induced Invitro Antibody Production in Humans: Tetanus Toxoid-Specific AntibodySynthesis. J. Immunol., 129:107.

49. Nutman, T. B., Withers, A. S., Ottesen, E. A., 1985, In VitroParasite Antigen-Induced Antibody Responses in Human HelminthInfections, J. Immunol., 135:2794.

50. Ollson, L., Andreasen, R. B., Osta, A., Christensen, B., Biberfield,P., 1984, J. Exp. Ned., 159:537.

51. Nadler, L. M., Stasherko, P., Hardy, R., Pesando, J. M., Yunis, E.J., Schlossman, S. F., 1981, Human Immunology, 1:77.

52. Koprowski, H., Steplewski, Z., Heryln, D., Herlyn, M., 1978, Proc.Natl. Acad. Sci., 75:3405.

53. Dippold, W. G., Lloyd, K. O., Li LTC, et al., 1980, Proc. Natl.Acad. Sci., 77:6114.

54. Morgan, A. C., Galloway, D. R., Reisfield, R. A., 1981, Hybridoma,1:27.

55. Brown, J. P., Woodburn, R. G., Hart, C. E., Hellstrom, I.,Hellstrom, K. E., 1981, Natl. Acad. Sci., 78:539.

56. Muller, M., Zotter, S., Kemme, C., 1976, J. Nat. Cancer Inst.,56:295.

57. Tamana, M., Kajdos, A. H., Niedermeir, W., Durkin, W. J., Mestecky,J., 1981, Cancer, 47:2696.

58. Shoenfeld, Y., Schwartz, R. S., 1984, New Engl. J. Med., 311:1019.

59. Miller, R. A., Maloney, D. G., Levy, R. A., 1982, N. Eng. J. Med.,307:687.

60. Larson, S. M., Brown, J. P., Wright, P. W., et al., 1983, J. Nucl.Med., 4:123.

61. Miller, R. A., Maloney, D., Stratte, P., Levy, R., 1983, Hybridoma2:238.

62. Dillman, R. O., Shawler, D. L., Dillman, J. B., Royston, I., 1984,J. Clin. Oncol., 2:881.

63. Nowinski, R., Berglund, C., Lane, J., et al., 1980, Science,210:537.

64. Steinitz, M., Klein, G., Koshimies, S., Makel, C., 1977, Nature,269:420.

65. Lane, H. C., Shelhamer, J., Mostowski, H. S., Fauci, A. S., 1982, J.Exp. Med., 155:333.

66. Melchers, F., Potter, M., Warner, N. L., 1978, Current Top.Microbiol. and Immunol., 8:1X.

67. Houghton, A. N. Brooks, H., Cote, J., et al., 1983, J. Exp. Med.,158:53.

68. Abrams, P. G., Knost, J. A., Clarke, G. Wilburn, S., Oldham, R. K.,Foon, K. A., 1983, J. Immunol., 131:1201.

69. Popovic, M., Sarngadharan, M. G., Read E., Gallo R. C., 1984,Detection, Isolation, and Continuous Production of CytopathicRetroviruses (HTLV-III) form Patients with AIDS and Pre-AIDS. Sci224:497-500.

70. Kanki, P. J., M'Boup, S., Ricard, D., Barin, F., Denis, F., Boye,C., Sangare, L., Travers, K., Albaum, M., Marlink, R., Romet-Lemmonne,J. L., Essex, M., 1987, Human T-Lymphotropic Virus Type 4 and the HumanImmunodeficiency Virus in West Africa, Sci. 236:827-831.

71. Kaminsky, L. S., McHugh, T., Stites, D., Volberding, P., Henle G.,Henle, W., Levy, J. A., 1985, High Prevalence of Antibodies to AcquiredImmune Deficiency Syndrome (AIDS)-Associated Retrovirus (ARV) in AIDSand Related Conditions But Not in Other Disease States, P.N.A.S.,82:5535-5539.

72. Gallo, R. C., Salahuddin, S. Z., Popovic, M., Shearer, G. M.,Kaplan, M., Haynes, B. F., Palker, T. J., Redfield, R., Oleske, J.,Safai, B., White, G., Foster, P., Markham, P. D., 1984, FrequentDetection and Isolation of Cytopathic Retroviruses (HTLV-III) fromPatients with AIDS and at Risk for AIDS, Sci. 224:500-503.

73. Clavel, F., et al., 1986, Isolation of a New Human Retrovirus fromWest African Patients with AIDS. Sci. 233:343-346.

74. Mayer, K. H., et al., 1987, Correlation of Enzyme-LinkedImmunoabsorbent Assays for Serum Human Immunodeficiency Virus Antigenand Antibodies To Recombinant Viral Proteins with Subsequent ClinicalOutcomes in a Cohort of Asymptomatic Homosexual Men, Am. J. Med.83:208-212.

75. McDougal, J. S., et al. 1987, Antibody Response to HumanImmunodeficiency Virus in Homosexual Men, J. Clin, Inves. 80:316-324.

76. Zagury, D., et al, 1986, Long-Term Cultures of HTLV-III-Infected TCells: A Model of Cytopathology of T Cell Depletion in AIDS, Sci.231:850-853.

77. Sattentau, Q. J., et al., 1986, Epitopes of the CD4 Antigen and HIVInfection, Sci. 234:1120-1123.

78. Chanh, T. C., et al., 1987, Monoclonal Anti-Idiotypic AntibodyMimics the CD4 Receptor and Binds Human Immunodeficiency Virus, P.N.A.S.84:3891-3895.

79. McDougal, J. S., et al., 1986, Binding of the Human RetrovirusHTLV-III/LAV/ARC/HIV to the CD4 (T4) Molecule: Conformation Dependence,Epitope Mapping, Antibody Inhibition, and Potential for IdiotypicMimicry, M. Immuno. 137:2937-2944.

80. Lane, H. C., et al., 1983, Abnormalities of B-Cell Activation andImmunoregulation in Patients with the Acquired ImmunodeficiencySyndrome, N. Eng. J. Med., 309:453-458.

81. Zanetti, A. R., et al., 1986, Hepatitis B Vaccination of 113Hemophiliacs: Lower Antibody Response in Anti-LAV/HTLV-III-PositivePatients, Am. J. Hemoto. 23:339-345.

82. Yarchoan, R., et al., 1986, Mechanisms of B Cell Activation inPatients with Acquired Immunodeficiency Syndrome and Related Disorders,J. Clin. Inves., 78:439-447.

83. Martinez-Maza, O., et al., 1987, Infection with the HumanImmunodeficiency Virus (HIV) is Associated with An In Vivo Increase In BLymphocyte Activation and Immaturity, J. Immuno. 138:3720-3724.

84. Aman, P., et al., 1984, Epstein-Barr Virus Susceptibility of NormalHuman B Lymphocyte Populations, J. Exp. Med., 159:208-220.

85. Robert-Guroff, M., et al., 1987, HTLV-III Neutralizing AntibodyDevelopment in Transfusion-Dependent Seropositive Patients withB-Thalassemia, J. Immuno. 138:3731-3736.

86. Robert-Guroff, M., et al., 1985, HTLV-III-Neutralizing Antibodies inPatients with AIDS and AIDS-Related Complex, Nature 316:72-74.

87. Anderson, K. C., et al., 1986, Transfusion-Acquired HumanImmunodeficiency Virus Infection Among Immunocompromised Persons, Ann.Int. Med. 105:519-527.

88. Groopman, J. E. et al., 1987, Characterization of SerumNeutralization Response to the Human Immunodeficiency Virus (HIV), AidsRes. Hum. Retroviruses 3:71-85.

89. Weiss, R. A., 1985, Neutralization of Human T-Lymphotropic VirusType III by Sera of AIDS and AIDS-Risk Patients, Nature 316:69-71.

90. Laurence, J., et al., 1987, Characterization and TranscriptaseActivity, Sci. 235:1501-1504.

91. Lifson, J. D., 1986, Induction of CD4-Dependent Cell Fusion by theHTLV-III/LAV Envelope Glycoprotein, Nature 323:725-728.

92. Hahn, B. H., et al., 1986, Genetic Variation in HTLV-III/LAV OverTime in Patients with AIDS or at Risk for AIDS, Sci. 232:1548-1553.

93. Robert-Guroff, M., 1986, In Vitro Generation of an HTLV-III Variantby Neutralizing Antibody, J. Immuno. 137:3306-3309.

94. Wong-Staal, F., et al., 1985, Genomic Diversity of HumanT-Lymphotropic Virus Type III (HTLV-III) Sci. 229:759-762.

95. Ho, D. D., et al., 1987, Human Immunodeficiency Virus NeutralizingAntibodies Recognize Several Conserved Domains on the EnvelopeGlycoproteins, J. Virol. 61:2024-2028.

96. Cronin, W., et al., 1985, Anti-Lymphocyte Antibodies in Patientswith Acquired Immune Deficiency Syndrome (AIDS), Firs Int. Aids Mtg.,Atlanta.

97. Kiprov, D. D., et al., 1985, Correlation of AntilymphocyteAntibodies (ALA) with Seropositivity of Lymphadenopathy Virus (LAV).Firs Int. Aids Mtg., Atlanta.

98. Lowder, J. N., et al., 1987, Suppression of Anti-MouseImmunoglobulin Antibodies in Subhuman Primates Receiving MurineMonoclonal Antibodies Against T Cell Antigens, J. Immuno. 138:401-406.

99. Monroe, J. G., et al., 1986, Anti-Idiotypic Antibodies and Disease,Immuno. Inves. 15:263-286.

100. Wasserman, N. H. et al., 1982, P.N.A.S. 79:4810-4814.

101. Cleveland, W. L. et al., 1983, Nature, 305:56-57.

What is claimed:
 1. A method of inhibiting the binding of HIV-1 to humancells comprising administering to a human a human monoclonal antibody inan amount effective to inhibit said binding, wherein the monoclonalantibody is directed to an epitope on HIV-1, and which epitope isrecognized by monoclonal antibody F105 produced by the hybridomadesignated F105 (ATCC No. HB-9583).
 2. The method of claim 1 wherein thehuman monoclonal antibody is F105.